Hyaluronidase compositions and methods of using same for determining the etiology of peri-orbital puffiness

ABSTRACT

The present disclosure provides methods for determining an etiology of upper and/or lower eyelid puffiness by examining a subject with squinted eyes; and determining if upper and/or lower eyelid puffiness does not improve, improves, partially improves, or worsens, wherein the etiology of the upper and/or lower eyelid puffiness is diagnosed to be anterior to the orbicularis oculi muscle if the puffiness does not improve, wherein the etiology of the upper and/or lower eyelid puffiness is determined to be posterior to the orbicularis oculi muscle if the puffiness improves, wherein the etiology of the upper and/or lower eyelid puffiness is determined to be anterior and posterior to the orbicularis oculi muscle if the puffiness partially improves, or wherein the puffiness is determined to be secondary to hypertrophy of the orbicularis muscle or if the puffiness worsens.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claim priority to U.S. Provisional patentApplication Ser. No. 62/967,775, filed Jan. 30, 2020, which isincorporated herein by reference in its entirety.

FIELD

The present disclosure generally relates to methods for determining theetiology of peri-orbital puffiness.

BACKGROUND

The appearance of puffiness or fullness around the eyes (e.g., bagsunder the eyes) is a concern to millions of people particularly as theyage. It is generally believed that shifting forward (pseudoherniation)of fat pads within the orbit of the eye (two along the upper eyelid andthree along the lower eyelid) is the cause of this puffiness.

Consequently, a surgical procedure (blepharoplasty) is often performedwhich involves the removal of segments of these fat pads from theeyelids. This surgery carries risks such as excessive fat removalleading to a hollow appearance around the eyes, over-resection of theskin leading to difficulty closing the eyes and chronically dry eyes,potential visual compromise if there is bleeding that compresses theoptic nerve, and a longer recovery period. Also, if the condition ofpuffiness is not due to shifting of the eyelid fat pads, but rather dueto swelling of the soft tissues of the eyelids or swelling of the fatpads, surgery is the incorrect procedure and will not properly addressthe cause of puffiness or bags. As such, there exists a need for methodsto determine the etiology of peri-orbital puffiness.

SUMMARY

The present disclosure addresses the above need by providing a methodfor determining an etiology of upper and/or lower eyelid puffiness. Suchmethods may include examining a subject with squinted eyes (e.g., thesubject may be instructed to squint his or her eyes); and determining ifupper and/or lower eyelid puffiness does not improve, improves,partially improves, or worsens, wherein the etiology of the upper and/orlower eyelid puffiness is determined to be anterior to the orbicularisoculi muscle if the puffiness does not improve, wherein the etiology ofthe upper and/or lower eyelid puffiness is determined to be posterior tothe orbicularis oculi muscle if the puffiness improves, wherein theetiology of the upper and/or lower eyelid puffiness is determined to beanterior and posterior to the orbicularis oculi muscle if the puffinesspartially improves, or wherein the puffiness is determined to besecondary to hypertrophy of the orbicularis muscle or if the puffinessworsens.

In some embodiments of each or any of the above- or below-mentionedembodiments, the subject is in an upright position with head in aFrankfort horizontal plane.

In some embodiments of each or any of the above- or below-mentionedembodiments, the methods further comprise the step of instructing thesubject to squint or tighten the orbicularis oculi muscle.

In some embodiments of each or any of the above- or below-mentionedembodiments, the etiology of the upper and/or lower eyelid puffiness isdetermined to be anterior to the orbicularis oculi muscle, and whereinthe method further comprises administering a protein havinghyaluronidase activity into the soft tissue anterior to the orbicularisoculi muscle.

In some embodiments of each or any of the above- or below-mentionedembodiments, the etiology of the upper and/or lower eyelid puffiness isdetermined to be posterior to the orbicularis oculi muscle, and whereinthe method further comprises the step of determining if the upper and/orlower eyelid puffiness is secondary to pseudoherniation of upper and/orlower eyelid fat pads, edema of upper and/or lower eyelid fat pads, orupper and/or lower eyelid fat pad pseudoherniation and edema.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness of the lower eyelid fat pads are assessed byasking the subject to look straight up, look up and to the right, andlook up and to the left.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness of the lower eyelid fat pads is due topseudoherniation of the lower eyelid fat pads and surgery is indicatedif the lower eyelid fat pads protrude and are individually isolated.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness is due to pseudoherniation and edema of thelower eyelid fat pads if the lower eyelid fat pads protrude and are notindividually isolated.

In some embodiments of each or any of the above- or below-mentionedembodiments, a protein having hyaluronidase activity is injected intothe lower eyelid fat pads to determine the extent of edema of the lowereyelid fat pads.

In some embodiments of each or any of the above- or below-mentionedembodiments, the methods further comprise resecting a portion of thelower eyelid fat pads.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness of the upper eyelid fat pads are assessed byasking the subject to look straight down, look down and to the right,and look down and to the left.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness of the upper eyelid fat pads is due topseudoherniation of upper eyelid fat pads and surgery is indicated ifthe upper eyelid fat pads protrude and are individually isolated.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness is due to pseudoherniation and edema of uppereyelid fat pads if the upper eyelid fat pads protrude and are notindividually isolated.

In some embodiments of each or any of the above- or below-mentionedembodiments, a protein having hyaluronidase activity is injected intothe upper eyelid fat pads to determine the extent of edema of the eyelidfat pads.

In some embodiments of each or any of the above- or below-mentionedembodiments, the methods further comprise resecting a portion of theupper eyelid fat pads.

In some embodiments of each or any of the above- or below-mentionedembodiments, the etiology of the upper and/or lower eyelid puffiness isdetermined to be anterior and posterior to the orbicularis oculi muscle,and wherein the method further comprises injecting a protein havinghyaluronidase activity into the upper and/or lower eyelid fat pads.

In some embodiments of each or any of the above- or below-mentionedembodiments, the etiology of the upper and/or lower eyelid puffiness isdetermined to be anterior and posterior to the orbicularis oculi muscle,and wherein the method further comprises assessing whether the puffinessis partially due to pseudoherniation of eyelid fat pads or edema of thefat pads, or eyelid fat pad pseudoherniation and edema.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness of the lower eyelid fat pads are assessed byasking the subject to look straight up, look up and to the right, andlook up and to the left.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness is due to pseudoherniation of the lowereyelid fat pads and surgery is indicated if the lower eyelid fat padsprotrude and are individually isolated.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness is due to pseudoherniation and edema of thelower eyelid fat pads if the lower eyelid fat pads protrude and are notindividually isolated.

In some embodiments of each or any of the above- or below-mentionedembodiments, a protein having hyaluronidase activity is injected intothe lower eyelid fat pads to determine the extent of edema of the eyelidfat pads.

In some embodiments of each or any of the above- or below-mentionedembodiments, the methods further comprise resecting a portion of thelower eyelid fat pads.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness of the upper eyelid fat pads are assessed byasking the subject to look straight down, look down and to the right,and look down and to the left.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness of the upper eyelid fat pads is due topseudoherniation of the upper eyelid fat pads and surgery is indicatedif the upper eyelid fat pads protrude and are individually isolated.

In some embodiments of each or any of the above- or below-mentionedembodiments, the puffiness of the upper eyelid fat pads is due topseudoherniation and edema of the upper eyelid fat pads if the uppereyelid fat pads protrude and are not individually isolated.

In some embodiments of each or any of the above- or below-mentionedembodiments, a protein having hyaluronidase activity is injected intothe upper eyelid fat pads to determine the extent of edema of the uppereyelid fat pads.

In some embodiments of each or any of the above- or below-mentionedembodiments, the methods further comprise resecting a portion of theupper eyelid fat pads.

In some embodiments of each or any of the above- or below-mentionedembodiments, a neuromodulator is indicated if the puffiness isdetermined to be secondary to hypertrophy of the orbicularis muscle orif the puffiness worsens.

The present disclosure also provides a method for determining anetiology of peri-orbital puffiness by performing an eyelid squint test;and observing an impact of a movement of an orbicularis oculi muscle onprotrusion of eyelid fat pads, wherein the etiology of the upper and/orlower eyelid puffiness is determined to be anterior to the orbicularisoculi muscle if the puffiness does not improve, wherein the etiology ofthe upper and/or lower eyelid puffiness is determined to be posterior tothe orbicularis oculi muscle if the puffiness improves, wherein theetiology of the upper and/or lower eyelid puffiness is determined to beanterior and posterior to the orbicularis oculi muscle if the puffinesspartially improves, or wherein the puffiness is determined to besecondary to hypertrophy of the orbicularis muscle or if the puffinessworsens.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the disclosure,shown in the figures are embodiments which are presently preferred. Itshould be understood, however, that the disclosure is not limited to theprecise arrangements, examples and instrumentalities shown.

FIGS. 1A-1B show a patient seated in the Frankfort horizontal plane witheye puffiness before (Panel A) and 3 months after (Panel B) subcutaneousinjections of a total of 40 U of HYLENEX to four injection sitesincluding: the medial, central and lateral aspects of each of the lowereyelids and the lateral aspect of each of the upper eyelids (10 U persite).

FIGS. 2A and 2B show a patient seated in the Frankfort horizontal planewith eye puffiness before (Panel A) and 3 months after (Panel B)subcutaneous injections of a total of 40 U of HYLENEX to four injectionsites including: the medial, central and lateral aspects of each of thelower eyelids and the lateral aspect of each of the upper eyelids (10 Uper site).

FIGS. 3A and 3B show a patient seated in the Frankfort horizontal planewith eye puffiness before (Panel A) and approximately 1 month after(Panel B) subcutaneous injections of a total of 40 U of HYLENEX to fourinjection sites including: the medial, central and lateral aspects ofeach of the lower eyelids and the lateral aspect of each of the uppereyelids (10 U per site).

FIGS. 4A and 4B show a patient seated in the Frankfort horizontal planewith eye puffiness before (Panel A) and 2 months after (Panel B)subcutaneous injections of a total of 40 U of HYLENEX to four injectionsites including: the medial, central and lateral aspects of each of thelower eyelids and the lateral aspect of each of the upper eyelids (10 Uper site).

FIGS. 5A-5H show a patient seated in the Frankfort horizontal plane witheye puffiness before (Panel A; Aug. 15, 2019) and approximately 1 monthafter (Panel B; Sep. 4, 2019) subcutaneous injections of a total of 40 Uof HYLENEX to four injection sites including: the medial, central andlateral aspects of each of the lower eyelids and the lateral aspect ofeach of the upper eyelids (10 U per site). Panels C-H provide magnifiedimages of the patients' eyes before (Panels C, E, and G) and after(Panels D, F, and H) treatment with HYLENEX. Panels C and D depict amagnified view of the patient's right eye before and after treatmentwith HYLENEX, respectively, while asked to look straight ahead level tothe ground. Panels E and F depict a magnified view of the patient'sright eye before and after treatment with HYLENEX, respectively, whileasked to look upwards while maintaining her head in the Frankforthorizontal plane. Panels G and H depict a magnified view of thepatient's left eye before and after treatment with HYLENEX,respectively, while asked to look upwards while maintaining her head inthe Frankfort horizontal plane.

DETAILED DESCRIPTION

It is estimated that at least 32 million Americans over twenty-fiveyears of age suffer from periorbital puffiness or bags around theireyes. This condition is generally attributed to pseudoherniation of oneor more of the fat pads located around their eyes. Consequently, thesepatients often undergo a surgical blepharoplasty procedure to lessentheir extent of puffiness or bags by removing and/or repositioning thefat pads located around the eyes. However, many patients withperiorbital puffiness or bags do not have pseudoherniation of the fatpads located around their eyes. Instead, the puffiness or bags are dueto swelling (edema) of the tissues in that region. Given that puffinessmay be due to edema and/or a structural change (e.g., pseudoherniationof one or more fat pads) in the periorbital region there exists a needaccurately diagnose the etiology of the puffiness since unnecessarysurgery is both costly and may lead to unintended side effectsincluding, a patient exhibiting hollowness in the periorbital region.The inventors have developed a protocol that permits the targetedintervention of periorbital puffiness by diagnosing the etiology of theperiorbital puffiness (e.g., the etiology of the upper and/or lowereyelid puffiness is diagnosed to be anterior to the orbicularis oculimuscle if the puffiness does not improve, wherein the etiology of theupper and/or lower eyelid puffiness is diagnosed to be posterior to theorbicularis oculi muscle if the puffiness improves, wherein the etiologyof the upper and/or lower eyelid puffiness is diagnosed to be anteriorand posterior to the orbicularis oculi muscle if the puffiness partiallyimproves, or wherein the puffiness is diagnosed to be secondary tohypertrophy of the orbicularis muscle or if the puffiness worsens).Advantageously, such methods may be used to select an appropriatetreatment regimen for patients that exhibit peri-orbital puffiness.

Pseudoherniation of the eyelid fat pads is routinely determined byapplying gentle pressure along the globe to see if the fat padsprotrude. However, most people have some degree of fat protrusion withpressure along the globe even when only a minimal or no pseudoherniationexists. Furthermore, even if the puffiness is due to edema and notpseudoherniation of the fat pads, pressure on the globe will increasethe eyelid puffiness mimicking pseudoherniation. Consequently, theinventors have developed an examination algorithm to determine the mostprobable causes of peri-orbital puffiness and the most logical treatmentapproach that incorporates an Eyelid Squint Test (EST) and the impact ofthe movement of extraocular muscle (EOM) on the protrusion of the fatpads.

The EST begins with a patient in an upright position and head in theFrankfort horizontal plane. The patient is then asked to squint ortighten the orbicularis oculi muscle. The following observations anddeterminations in 1.), 2.), 3.), and/or 4.) below are then made:

1. If the periorbital puffiness persists or does not improve, theetiology is anterior to the orbicularis oculi muscle. The periorbitalpuffiness is determined to be due to soft tissue edema and the subjectis indicated for treatment with a hyaluronidase injection into the softtissues anterior to the orbicularis oculi muscle.

2. If the puffiness disappears (or improves), the etiology of thepuffiness is determined to be posterior to the orbicularis oculi muscle.As such, the puffiness may be secondary to pseudoherniation of the fatpads or edema of the fat pads, or a combination of fat padpseudoherniation and edema. Consequently, the steps in i) and ii) beloware performed to ascertain the cause of the puffiness and theappropriate course of treatment.

-   -   i. While maintaining the head in the Frankfort horizontal plane,        the subject is then asked to look straight up, look up and to        the right, and look up and to the left        -   1. If the lower lid fat pads protrude and are individually            isolated, the puffiness is due to pseudoherniation of the            fat pads. Surgery is indicated to remove all or a portion of            the fat pads.        -   2. If the lower lid fat pads protrude and are not            individually isolated, the puffiness is due to            pseudoherniation and edema of the fat pads.            -   a. Hyaluronidase may be injected into the fat pads first                to determine the extent of edema in the fat pads so as                to minimize the risk of over resection of the fat pads                during surgery.    -   ii. While maintaining the head in the Frankfort horizontal        plane, the subject is asked to look straight down, look down and        to the right, and look down and to the left        -   1. If the upper lid fat pads protrude and are individually            isolated, the puffiness is due to pseudoherniation of the            fat pads. Surgery is indicated to remove all or a portion of            the fat pads.        -   2. If the upper lid fat pads protrude and are not            individually isolated, the puffiness is due to            pseudoherniation and edema of the fat pads.            -   a. Hyaluronidase is injected into the fat pads first to                determine the extent of edema in the fat pads so as to                minimize the risk of over resection of the fat pads                during surgery.

3. If the periorbital puffiness only partially improves, the etiology isdue to processes both anterior and posterior to the orbicularis oculimuscle. As such, the puffiness is determined to be partially due to softtissue edema anterior to the orbicularis oculi muscle, and the subjectwill have some improvement from a hyaluronidase injection into thesesoft tissues. Additionally, the puffiness is also partially due topseudoherniation of the fat pads or edema of the fat pads, or acombination of fat pad pseudoherniation and edema. Consequently, thesteps in i) and ii) below are performed to ascertain the cause of thepuffiness and the appropriate course of treatment.

-   -   i. While maintaining the head in the Frankfort horizontal plane,        the subject is asked to look straight up, look up and to the        right, and look up and to the left        -   1. If the lower lid fat pads protrude and are individually            isolated, the puffiness is due to pseudoherniation of the            fat pads. Surgery is indicated to remove all or a portion of            the fat pads.        -   2. If the lower lid fat pads protrude and are not            individually isolated, the puffiness is due to            pseudoherniation and edema of the fat pads.            -   a. Hyaluronidase may be injected into the fat pads first                to determine the extent of edema in the fat pads so as                to minimize the risk of over resection of the fat pads                during surgery.    -   ii. While maintaining the head in the Frankfort horizontal        plane, the subject is asked to look straight down, look down and        to the right, and look down and to the left        -   1. If the upper lid fat pads protrude and are individually            isolated, the puffiness is due to pseudoherniation of the            fat pads. Surgery is indicated to remove all or a portion of            the fat pads.        -   2. If the upper lid fat pads protrude and are not            individually isolated, the puffiness is due to            pseudoherniation and edema of the fat pads.            -   a. Hyaluronidase is injected into the fat pads first to                determine the extent of edema in the fat pads so as to                minimize the risk of over resection of the fat pads                during surgery.

4. If the periorbital puffiness worsens, it is secondary to hypertrophyof the orbicularis oculi muscle and a neuromodulator may be indicatedfor treatment.

As used herein, the term “periorbital puffiness” also known as swellingor puffiness around the eyes, is the appearance of swelling in thetissues around the eyes, called the orbits. It is almost exclusivelycaused by fluid buildup around the eyes, or periorbital edema.

As used herein, “hyaluronidase” refers to an enzyme that degradeshyaluronic acid. Hyaluronidases include bacterial hyaluronidases (EC4.2.99.1), hyaluronidases from leeches, spiders, snakes, parasites, andcrustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC3.2.1.35). Hyaluronidases also include any of non-human originincluding, but not limited to, murine, canine, feline, leporine, avian,bovine, ovine, porcine, equine, piscine, ranine, bacterial, and any fromleeches, other parasites, and crustaceans. Hyaluronidases also includethose of human origin. Also included amongst hyaluronidases are solublehyaluronidases.

Reference to hyaluronidases includes precursor hyaluronidasepolypeptides and mature hyaluronidase polypeptides (such as those inwhich a signal sequence has been removed), truncated forms thereof thathave activity, and includes allelic variants and species variants,variants encoded by splice variants, and other variants. Hyaluronidasesalso include those that contain chemical or posttranslationalmodifications and those that do not contain chemical orposttranslational modifications. Such modifications include, but are notlimited to, pegylation, albumination, glycosylation, farnesylation,carboxylation, hydroxylation, phosphorylation, and other polypeptidemodifications known in the art.

As used herein, a soluble hyaluronidase refers to a polypeptidecharacterized by its solubility under physiologic conditions. Solublehyaluronidases can be distinguished, for example, by its partitioninginto the aqueous phase of a Triton X-114 solution warmed to 37° C.(Bordier et al., (1981) J. Biol. Chem., 256:1604-7). Membrane-anchored,such as lipid anchored hyaluronidases, will partition into the detergentrich phase, but will partition into the detergent-poor or aqueous phasefollowing treatment with Phospholipase-C. Included among solublehyaluronidases are membrane anchored hyaluronidases in which one or moreregions associated with anchoring of the hyaluronidase to the membranehas been removed or modified, where the soluble form retainshyaluronidase activity. Soluble hyaluronidases include recombinantsoluble hyaluronidases and those contained in or purified from naturalsources, such as, for example, testes extracts from sheep or cows.

As used herein, the term “periorbital puffiness” also known as swellingor fullness around the eyes is the appearance of swelling in the tissuesaround the eyes, called the orbits. It may be caused by fluid builduparound the eyes, or periorbital edema including edema in theperi-orbital fat pads and soft tissues. Periorbital puffiness may alsobe due to swelling or fullness of the malar region.

As used herein, “hyaluronidase activity” refers to the ability of aprotein to cleave hyaluronic acid. In vitro assays to determine thehyaluronidase activity of hyaluronidases are known in the art anddescribed herein. Exemplary assays include the microturbidity assay thatmeasures cleavage of hyaluronic acid by hyaluronidase indirectly bydetecting the insoluble precipitate formed when the uncleaved hyaluronicacid binds with serum albumin.

The terms, “treating” or “treatment” of a disease, disorder, orcondition includes at least partially: (1) preventing the disease,disorder, or condition, i.e. causing the clinical symptoms of thedisease, disorder, or condition not to develop in a mammal that isexposed to or predisposed to the disease, disorder, or condition butdoes not yet experience or display symptoms of the disease, disorder, orcondition; (2) inhibiting the disease, disorder, or condition, i.e.,arresting or reducing the development of the disease, disorder, orcondition or its clinical symptoms; or (3) relieving the disease,disorder, or condition, i.e., causing regression of the disease,disorder, or condition or its clinical symptoms. The term “treating,”includes to reducing any detectable amount or eliminating in anindividual puffiness. In some embodiments, puffiness may be reduced atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90% or at least about 100%.

The terms “prevention”, “prevent”, “preventing”, “suppression”,“suppress”, “suppressing”, “inhibit” and “inhibition” as used hereinrefer to a course of action initiated in a manner so as to prevent,suppress or reduce, either temporarily or permanently, the onset of aclinical manifestation of the disease state or condition. Suchpreventing, suppressing or reducing need not be absolute to be useful.

The terms “improvement” or “improving” as used herein in reference toperiorbital puffiness refers to a reduction in periorbital puffiness.

The terms “reducing” or “reduction” as used herein refers to a decrease(or lowering) in the amount, mass, and/or volume of periorbitalpuffiness. Such reduction can be measured and determined by measuringthe amount of puffiness according to one or more of the methodsdescribed herein including, for example, at an initial time point priorto the administering of the compounds described herein and thenmeasuring the amount of puffiness at various time points (e.g. duringthe period of administering the compounds described herein as well afterthe administering has ceased). For example, a subject's puffiness can bemeasured prior to beginning a treatment regimen with the compoundsdescribed herein and then measured during and after the treatmentregimen. A decrease in puffiness is indicative of a reduction inpuffiness. Additionally, the reduction of puffiness can be determinedqualitatively such as by photographing the face or eyes, at various timepoints before, during, and after a treatment regimen where the reductionin puffiness can be determined by visual inspection of the images. Areduction in puffiness includes, for example, a 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or greater loweringor decrease in the amount, mass, and/or volume of periorbital puffiness.Alternatively, a subject's puffiness is given a score of 10 (on a scaleof 1 to 10 with 1 being no periorbital puffiness) prior to treatmentwith the composition disclosed herein. The subject and/or medicalpractitioner that administered the composition then scores the puffinessafter treatment with a subjective grade based on the original score of10 (e.g., the score is given based on a visual assessment of the subjectfrom a before photograph of the periorbital puffiness). A decrease inpuffiness measured as a score of 6 or less indicates a reduction inperiorbital puffiness. Alternatively, a reduction in puffiness may bedetermined by a reduction in in a PFAS grade (e.g., a score of L 0/3E[left eye; upper eyelid over lower eyelid] and a R 0/3E [right eye;upper eyelid over lower eyelid] to a score of L 0/1E and L 0/1E). Areduction in PFAS grade corresponds to a reduction in the Grade number(e.g., a 3 to a 2).

The terms “partially improve” or partial improvement” as used herein inreference to periorbital puffiness refers to a small (or minor)reduction in periorbital puffiness such as 5%, 10%, 15%, 20%, or 25%lowering or decrease in the amount, mass, and/or volume of puffiness.Alternatively, a subject's puffiness is given a score of 10 (on a scaleof 1 to 10 with 1 being no periorbital puffiness) prior to treatmentwith the composition disclosed herein. The subject and/or medicalpractitioner that administered the composition then scores the puffinessafter treatment with a subjective grade based on the original score of10 (e.g., the score is given based on a visual assessment of the subjectfrom a before photograph of the periorbital puffiness). A decrease inpuffiness measured as a score of 7 to 9 indicates a partial improvementin periorbital puffiness.

The terms “worsens” or “worse” as used herein in reference toperiorbital puffiness refer to an increase in periorbital puffiness.

The terms “increased” or “increase” as used herein refers to an increasein the amount, mass, and/or volume of periorbital puffiness. Suchincrease can be measured and determined by measuring the amount ofpuffiness according to one or more of the methods described hereinincluding, for example, at an initial time point prior to theadministering of the compounds described herein and then measuring theamount of puffiness at various time points (e.g. during the period ofadministering the compounds described herein as well after theadministering has ceased). For example, a subject's puffiness can bemeasured prior to beginning a treatment regimen with the compoundsdescribed herein and then measured during and after the treatmentregimen. Additionally, the increase in puffiness can be determinedqualitatively such as by photographing the face or eyes including, forexample, at various time points before, during, and after a treatmentregimen where the reduction in puffiness can be determined by visualinspection of the images. An increase in puffiness incudes, for example,a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100%, or increase in the amount, mass, and/orvolume of periorbital puffiness. Alternatively, an increase in puffinessmay be determined by an increase in a PFAS grade (e.g., a score of L0/1E [left eye; upper eyelid over lower eyelid] and a R 0/1E [right eye;upper eyelid over lower eyelid] to a score of L 0/3E and L 0/3E). Aincrease in PFAS grade corresponds to an increase in the Grade number(e.g., a 1 to a 3).

As used herein, the term “subject” refers to an animal, including amammal, such as a human being.

As used herein, a patient refers to a human subject.

As used herein, amelioration of the symptoms by a treatment, such as byadministration of a pharmaceutical composition or other therapeutic,refers to any lessening, whether permanent or temporary, lasting ortransient, of the symptoms that can be attributed to or associated withadministration of the composition or therapeutic.

As used herein, prevention or prophylaxis refers to methods in which therisk of developing disease or condition is reduced.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect. Hence, it is thequantity necessary for preventing, curing, ameliorating, arresting orpartially arresting a symptom of a disease, disorder, or condition.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a compound, comprising “an extracellular domain”includes compounds with one or a plurality of extracellular domains.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.”

Hyaluronidase

Hyaluronidases are a family of enzymes that degrade hyaluronic acid.There are three general classes of hyaluronidases; mammalianhyaluronidase, bacterial hyaluronidase and hyaluronidase from leeches,other parasites and crustaceans. Mammalian-type hyaluronidases (EC3.2.1.35) are endo-β-N-acetyl-hexosaminidases that hydrolyze the β1→4glycosidic bond of hyaluronan into various oligosaccharide lengths suchas tetrasaccharides and hexasaccharides. They have both hydrolytic andtransglycosidase activities, and can degrade hyaluronan and chondroitinsulfates (CS), generally C4-S and C6-S. Hyaluronidases of this typeinclude, but are not limited to, hyaluronidases from cows (bovine),mouse, pig, rat, rabbit, sheep (ovine), orangutan, cynomolgus monkey,guinea pig, and human hyaluronidases.

Mammalian hyaluronidases can be further subdivided into those that areneutral active, predominantly found in testes extracts, and acid active,predominantly found in organs such as the liver. Exemplary neutralactive hyaluronidases include PH20. Human PH20 (also known as SPAM1 orsperm surface protein PH20), is generally locked to the plasma membranevia a glycosylphosphatidyl inositol (GPI) anchor. It is naturallyinvolved in sperm-egg adhesion and aids penetration by sperm of thelayer of cumulus cells by digesting hyaluronic acid. Alignment of bovinePH20 with the human PH20 shows only weak homology, with multiple gapsexisting from amino acid 470 through to the respective carboxy terminidue to the absence of a GPI anchor in the bovine polypeptide (see e.g.,Frost GI (2007) Expert Opin. Drug. Deliv. 4: 427-440). In fact, no clearGPI anchor is predicted in any other PH20 species besides humans. Thus,PH20 polypeptides produced from ovine and bovine exist as soluble forms.Though bovine PH20 exists very loosely attached to the plasma membrane,it is not anchored via a phospholipase sensitive anchor (Lalancette etal, Biol Reprod. 2001 August; 65(2):628-36.). This unique feature ofbovine hyaluronidase has permitted the use of the soluble bovine testeshyaluronidase enzyme as an extract for clinical use (Wydase™, Hyalase™).

Besides human PH20 (also termed SPAM1), five hyaluronidase-like geneshave been identified in the human genome, HYAL1, HYAL2, HYAL3, HYAL4 andHYALP1. HYALP1 is a pseudogene, and HYAL3 has not been shown to possessenzyme activity toward any known substrates. The hyaluronidase-likeenzymes can also be characterized by those which are generally locked tothe plasma membrane via a glycosylphosphatidyl inositol anchor such ashuman HYAL2 and human PH20 (Danilkovitch-Miagkova, et al. (2003) ProcNatl Acad Sci USA. 100(8):4580-5), and those which are generally solublesuch as human HYAL1 (Frost et al, (1997) Biochem Biophys Res Commun.236(1):10-5).

In a preferred embodiment, the hyaluronidase is HYLENEX (having theamino acid sequence as set forth in SEQ ID NO: 1).

Glycosylation, including N- and O-linked glycosylation, of somehyaluronidases can be very important for their catalytic activity andstability. While altering the type of glycan modifying a glycoproteincan have dramatic effects on a protein's antigenicity, structuralfolding, solubility, and stability, most enzymes are not thought torequire glycosylation for optimal enzyme activity. Such hyaluronidasesare unique in this regard, in that removal of N-linked glycosylation canresult in near complete inactivation of the hyaluronidase activity. Forsuch hyaluronidases, the presence of N-linked glycans is critical forgenerating an active enzyme.

N-linked oligosaccharides fall into several major types (oligomannose,complex, hybrid, sulfated), all of which have (Man)3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn residuesthat fall within-Asn-Xaa-Thr/Ser-sequences (where Xaa is not Pro).Glycosylation at an-Asn-Xaa-Cys-site has been reported for coagulationprotein C. In some instances, the hyaluronidase can contain bothN-glycosidic and 0-glycosidic linkages.

Soluble hyaluronidases include any that exist in soluble form,including, but not limited to, Hyal1, bovine PH20 and ovine PH20,allelic variants thereof and other variants. Also included among solublehyaluronidase are any hyaluronidase that has been modified to besoluble. For example, human PH20, which is normally membrane anchoredvia a GPI anchor, can be made soluble by truncation of and removal ofall or a portion of the GPI anchor at the C-terminus. Solublehyaluronidases also include neutral active and acid activehyaluronidases, however, neutral active hyaluronidases are contemplatedfor use herein for purposes of subcutaneous administration.

Polypeptides of a soluble hyaluronidase set forth herein, can beobtained by methods well known in the art for protein purification andrecombinant protein expression. Any method known to those of skill inthe art for identification of nucleic acids that encode desired genescan be used. Any method available in the art can be used to obtain afull length (i.e., encompassing the entire coding region) cDNA orgenomic DNA clone encoding a hyaluronidase, such as from a cell ortissue source. Modified or variant soluble hyaluronidases, can beengineered from a wild type polypeptide, such as by site-directedmutagenesis.

Polypeptides can be cloned or isolated using any available methods knownin the art for cloning and isolating nucleic acid molecules. Suchmethods include PCR amplification of nucleic acids and screening oflibraries, including nucleic acid hybridization screening,antibody-based screening and activity-based screening.

Methods for amplification of nucleic acids can be used to isolatenucleic acid molecules encoding a desired polypeptide, including forexample, polymerase chain reaction (PCR) methods. A nucleic acidcontaining material can be used as a starting material from which adesired polypeptide-encoding nucleic acid molecule can be isolated. Forexample, DNA and mRNA preparations, cell extracts, tissue extracts,fluid samples (e.g. blood, serum, saliva), samples from healthy and/ordiseased subjects can be used in amplification methods. Nucleic acidlibraries also can be used as a source of starting material. Primers canbe designed to amplify a desired polypeptide. For example, primers canbe designed based on expressed sequences from which a desiredpolypeptide is generated. Primers can be designed based onback-translation of a polypeptide amino acid sequence. Nucleic acidmolecules generated by amplification can be sequenced and confirmed toencode a desired polypeptide.

Additional nucleotide sequences can be joined to a polypeptide-encodingnucleic acid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a polypeptide-encoding nucleicacid molecule. Examples of such sequences include, but are not limitedto, promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences, for example heterologous signalsequences, designed to facilitate protein secretion. Such sequences areknown to those of skill in the art. Additional nucleotide residuessequences such as sequences of bases specifying protein binding regionsalso can be linked to enzyme-encoding nucleic acid molecules. Suchregions include, but are not limited to, sequences of residues thatfacilitate or encode proteins that facilitate uptake of an enzyme intospecific target cells, or otherwise alter pharmacokinetics of a productof a synthetic gene. For example, enzymes can be linked to PEG moieties.

In addition, tags or other moieties can be added, for example, to aid indetection or affinity purification of the polypeptide. For example,additional nucleotide residues sequences such as sequences of basesspecifying an epitope tag or other detectable marker also can be linkedto enzyme-encoding nucleic acid molecules. Exemplary of such sequencesinclude nucleic acid sequences encoding a His tag (e.g., 6×His) or FlagTag.

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.).

Other expression vectors include the HZ24 expression vector exemplifiedherein. The insertion into a cloning vector can, for example, beaccomplished by ligating the DNA fragment into a cloning vector whichhas complementary cohesive termini. Insertion can be effected using TOPOcloning vectors (INVITROGEN, Carlsbad, Calif.). If the complementaryrestriction sites used to fragment the DNA are not present in thecloning vector, the ends of the DNA molecules can be enzymaticallymodified. Alternatively, any site desired can be produced by ligatingnucleotide sequences (linkers) onto the DNA termini; these ligatedlinkers can contain specific chemically synthesized oligonucleotidesencoding restriction endonuclease recognition sequences. In analternative method, the cleaved vector and protein gene can be modifiedby homopolymeric tailing. Recombinant molecules can be introduced intohost cells via, for example, transformation, transfection, infection,electroporation and sonoporation, so that many copies of the genesequence are generated.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

For recombinant expression of one or more of the desired proteins, suchas any described herein, the nucleic acid containing all or a portion ofthe nucleotide sequence encoding the protein can be inserted into anappropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedprotein coding sequence. The necessary transcriptional and translationalsignals also can be supplied by the native promoter for enzyme genes,and/or their flanking regions.

Also provided are vectors that contain a nucleic acid encoding theenzyme. Cells containing the vectors also are provided. The cellsinclude eukaryotic and prokaryotic cells, and the vectors are anysuitable for use therein.

Prokaryotic and eukaryotic cells, including endothelial cells,containing the vectors are provided. Such cells include bacterial cells,yeast cells, fungal cells, Archea, plant cells, insect cells and animalcells. The cells are used to produce a protein thereof by growing theabove-described cells under conditions whereby the encoded protein isexpressed by the cell, and recovering the expressed protein. Forpurposes herein, for example, the enzyme can be secreted into themedium.

Also provided are vectors that contain a sequence of nucleotides thatencodes the soluble hyaluronidase polypeptide coupled to the native orheterologous signal sequence, as well as multiple copies thereof. Thevectors can be selected for expression of the enzyme protein in the cellor such that the enzyme protein is expressed as a secreted protein.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding protein, or domains,derivatives, fragments or homologs thereof, can be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins can be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for a desired protein. Promoterswhich can be used include but are not limited to the SV40 early promoter(Bernoist and Chambon, Nature 290:304-310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), theregulatory sequences of the metallothionein gene (Brinster et al.,Nature 296:39-42 (1982)); prokaryotic expression vectors such as theβ-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA80:21-25 (1983)); see also “Useful Proteins from Recombinant Bacteria”:in Scientific American 242:79-94 (1980)); plant expression vectorscontaining the nopaline synthetase promoter (Herrara-Estrella et al.,Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNApromoter (Garder et al., Nucleic Acids Res. 9:2871 (1981)), and thepromoter of the photosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elementsfrom yeast and other fungi such as the Ga14 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7:425-515 (1987)); insulin gene control region which isactive in pancreatic beta cells (Hanahan et al., Nature 315:115-122(1985)), immunoglobulin gene control region which is active in lymphoidcells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444(1987)), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell45:485-495 (1986)), albumin gene control region which is active in liver(Pinckert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., Mol.Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)),alpha-1 antitrypsin gene control region which is active in liver (Kelseyet al., Genes and Devel. 1:161-171 (1987)), beta globin gene controlregion which is active in myeloid cells (Magram et al., Nature315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basicprotein gene control region which is active in oligodendrocyte cells ofthe brain (Readhead et al., Cell 48:703-712 (1987)), myosin lightchain-2 gene control region which is active in skeletal muscle (Shani,Nature 314:283-286 (1985)), and gonadotrophic releasing hormone genecontrol region which is active in gonadotrophs of the hypothalamus(Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a desired protein, or adomain, fragment, derivative or homolog, thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pQEexpression vectors (available from Qiagen, Valencia, Calif.; see alsoliterature published by Qiagen describing the system). pQE vectors havea phage T5 promoter (recognized by E. coli RNA polymerase) and a doublelac operator repression module to provide tightly regulated, high-levelexpression of recombinant proteins in E. coli, a synthetic ribosomalbinding site (RBS II) for efficient translation, a 6×His tag codingsequence, t0 and T1 transcriptional terminators, ColE1 origin ofreplication, and a beta-lactamase gene for conferring ampicillinresistance. The pQE vectors enable placement of a 6×His tag at eitherthe N- or C-terminus of the recombinant protein. Such plasm ids includepQE 32, pQE 30, and pQE 31 which provide multiple cloning sites for allthree reading frames and provide for the expression of N-terminally6×His-tagged proteins. Other exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pETexpression vectors (see, U.S. Pat. No. 4,952,496; available fromNOVAGEN, Madison, Wis.; see, also literature published by Novagendescribing the system). Such plasmids include pET 11 a, which containsthe T7lac promoter, T7 terminator, the inducible E. coli lac operator,and the lac repressor gene; pET 12a-c, which contains the T7 promoter,T7 terminator, and the E. coli ompT secretion signal; and pET 15b andpET19b (NOVAGEN, Madison, Wis.), which contain a His-Tag™ leadersequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn, the T7-lac promoter region and the T7 terminator.

Soluble hyaluronidase polypeptides can be produced by any method knownto those of skill in the art including in vivo and in vitro methods.Desired proteins can be expressed in any organism suitable to producethe required amounts and forms of the proteins, such as for example,needed for administration and treatment. Expression hosts includeprokaryotic and eukaryotic organisms such as E. coli, yeast, plants,insect cells, mammalian cells, including human cell lines and transgenicanimals. Expression hosts can differ in their protein production levelsas well as the types of post-translational modifications that arepresent on the expressed proteins. The choice of expression host can bemade based on these and other factors, such as regulatory and safetyconsiderations, production costs and the need and methods forpurification.

Many expression vectors are available and known to those of skill in theart and can be used for expression of proteins. The choice of expressionvector will be influenced by the choice of host expression system. Ingeneral, expression vectors can include transcriptional promoters andoptionally enhancers, translational signals, and transcriptional andtranslational termination signals. Expression vectors that are used forstable transformation typically have a selectable marker which allowsselection and maintenance of the transformed cells. In some cases, anorigin of replication can be used to amplify the copy number of thevector.

Soluble hyaluronidase polypeptides also can be utilized or expressed asprotein fusions. For example, an enzyme fusion can be generated to addadditional functionality to an enzyme. Examples of enzyme fusionproteins include, but are not limited to, fusions of a signal sequence,a tag such as for localization, e.g. a his6 tag or a myc tag, or a tagfor purification, for example, a GST fusion, and a sequence fordirecting protein secretion and/or membrane association.

Prokaryotes, especially E. coli, provide a system for producing largeamounts of proteins. Transformation of E. coli is simple and rapidtechnique well known to those of skill in the art. Expression vectorsfor E. coli can contain inducible promoters, such promoters are usefulfor inducing high levels of protein expression and for expressingproteins that exhibit some toxicity to the host cells. Examples ofinducible promoters include the lac promoter, the trp promoter, thehybrid tac promoter, the T7 and SP6 RNA promoters and the temperatureregulated APL promoter.

Proteins, such as any provided herein, can be expressed in thecytoplasmic environment of E. coli. The cytoplasm is a reducingenvironment and for some molecules, this can result in the formation ofinsoluble inclusion bodies. Reducing agents such as dithiothreitol andβ-mercaptoethanol and denaturants, such as guanidine-HCl and urea can beused to resolubilize the proteins. An alternative approach is theexpression of proteins in the periplasmic space of bacteria whichprovides an oxidizing environment and chaperonin-like and disulfideisomerases and can lead to the production of soluble protein. Typically,a leader sequence is fused to the protein to be expressed which directsthe protein to the periplasm. The leader is then removed by signalpeptidases inside the periplasm. Examples of periplasmic-targetingleader sequences include the pelB leader from the pectate lyase gene andthe leader derived from the alkaline phosphatase gene. In some cases,periplasmic expression allows leakage of the expressed protein into theculture medium. The secretion of proteins allows quick and simplepurification from the culture supernatant. Proteins that are notsecreted can be obtained from the periplasm by osmotic lysis. Similar tocytoplasmic expression, in some cases proteins can become insoluble anddenaturants and reducing agents can be used to facilitate solubilizationand refolding. Temperature of induction and growth also can influenceexpression levels and solubility, typically temperatures between 25° C.and 37° C. are used. Typically, bacteria produce aglycosylated proteins.Thus, if proteins require glycosylation for function, glycosylation canbe added in vitro after purification from host cells.

Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are wellknown yeast expression hosts that can be used for production ofproteins, such as any described herein. Yeast can be transformed withepisomal replicating vectors or by stable chromosomal integration byhomologous recombination. Typically, inducible promoters are used toregulate gene expression. Examples of such promoters include GAL1, GALTand GALS and metallothionein promoters, such as CUP1, AOX1 or otherPichia or other yeast promoter. Expression vectors often include aselectable marker such as LEU2, TRP1, HIS3 and URA3 for selection andmaintenance of the transformed DNA. Proteins expressed in yeast areoften soluble. Co-expression with chaperonins such as Bip and proteindisulfide isomerase can improve expression levels and solubility.Additionally, proteins expressed in yeast can be directed for secretionusing secretion signal peptide fusions such as the yeast mating typealpha-factor secretion signal from Saccharomyces cerevisae and fusionswith yeast cell surface proteins such as the Aga2p mating adhesionreceptor or the Arxula adeninivorans glucoamylase. A protease cleavagesite such as for the Kex-2 protease, can be engineered to remove thefused sequences from the expressed polypeptides as they exit thesecretion pathway. Yeast also is capable of glycosylation atAsn-X-Ser/Thr motifs.

Insect cells, particularly using baculovirus expression, are useful forexpressing polypeptides such as hyaluronidase polypeptides. Insect cellsexpress high levels of protein and are capable of most of thepost-translational modifications used by higher eukaryotes. Baculovirushave a restrictive host range which improves the safety and reducesregulatory concerns of eukaryotic expression. Typical expression vectorsuse a promoter for high level expression such as the polyhedrin promoterof baculovirus. Commonly used baculovirus systems include thebaculoviruses such as Autographa californica nuclear polyhedrosis virus(AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and aninsect cell line such as Sf9 derived from Spodoptera frugiperda,Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1). For high-levelexpression, the nucleotide sequence of the molecule to be expressed isfused immediately downstream of the polyhedrin initiation codon of thevirus. Mammalian secretion signals are accurately processed in insectcells and can be used to secrete the expressed protein into the culturemedium. In addition, the cell lines Pseudaletia unipuncta (A7S) andDanaus plexippus (DpN1) produce proteins with glycosylation patternssimilar to mammalian cell systems.

An alternative expression system in insect cells is the use of stablytransformed cells. Cell lines such as the Schneider 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

Mammalian expression systems can be used to express proteins includingsoluble hyaluronidase polypeptides. Expression constructs can betransferred to mammalian cells by viral infection such as adenovirus orby direct DNA transfer such as liposomes, calcium phosphate,DEAE-dextran and by physical means such as electroporation andmicroinjection. Expression vectors for mammalian cells typically includean mRNA cap site, a TATA box, a translational initiation sequence (Kozakconsensus sequence) and polyadenylation elements. IRES elements also canbe added to permit bicistronic expression with another gene, such as aselectable marker. Such vectors often include transcriptionalpromoter-enhancers for high-level expression, for example the SV40promoter-enhancer, the human cytomegalovirus (CMV) promoter and the longterminal repeat of Rous sarcoma virus (RSV). These promoter-enhancersare active in many cell types. Tissue and cell-type promoters andenhancer regions also can be used for expression.

Exemplary promoter/enhancer regions include, but are not limited to,those from genes such as elastase I, insulin, immunoglobulin, mousemammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin,beta globin, myelin basic protein, myosin light chain 2, andgonadotropic releasing hormone gene control. Selectable markers can beused to select for and maintain cells with the expression construct.Examples of selectable marker genes include, but are not limited to,hygromycin B phosphotransferase, adenosine deaminase, xanthine-guaninephosphoribosyl transferase, aminoglycoside phosphotransferase,dihydrofolate reductase (DHFR) and thymidine kinase. For example,expression can be performed in the presence of methotrexate to selectfor only those cells expressing the DHFR gene. Fusion with cell surfacesignaling molecules such as TCR-ζ and FcεRI-γ can direct expression ofthe proteins in an active state on the cell surface.

Many cell lines are available for mammalian expression including mouse,rat human, monkey, chicken and hamster cells. Exemplary cell linesinclude but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NSO(nonsecreting) and other myeloma cell lines, hybridoma andheterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are availableadapted to serum-free media which facilitates purification of secretedproteins from the cell culture media. Examples include CHO-S cells(Invitrogen, Carlsbad, Calif., cat #11619-012) and the serum free EBNA-1cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-42.). Celllines also are available that are adapted to grow in special mediumsoptimized for maximal expression. For example, DG44 CHO cells areadapted to grow in suspension culture in a chemically defined, animalproduct-free medium.

Method for purification of polypeptides, including soluble hyaluronidasepolypeptides or other proteins, from host cells will depend on thechosen host cells and expression systems. For secreted molecules,proteins are generally purified from the culture media after removingthe cells. For intracellular expression, cells can be lysed and theproteins purified from the extract. When transgenic organisms such astransgenic plants and animals are used for expression, tissues or organscan be used as starting material to make a lysed cell extract.Additionally, transgenic animal production can include the production ofpolypeptides in milk or eggs, which can be collected, and if necessary,the proteins can be extracted and further purified using standardmethods in the art.

Proteins, such as soluble hyaluronidase polypeptides, can be purifiedusing standard protein purification techniques known in the artincluding but not limited to, SDS-PAGE, size fraction and size exclusionchromatography, ammonium sulfate precipitation and ionic exchangechromatography, such as anion exchange. Affinity purification techniquesalso can be utilized to improve the efficiency and purity of thepreparations. For example, antibodies, receptors and other moleculesthat bind hyaluronidase enzymes can be used in affinity purification.Expression constructs also can be engineered to add an affinity tag to aprotein such as a myc epitope, GST fusion or His6 and affinity purifiedwith myc antibody, glutathione resin and Ni-resin, respectively. Puritycan be assessed by any method known in the art including gelelectrophoresis and staining and spectrophotometric techniques.

Hyaluronidase activity can be assessed using methods well known in theart. In one example, activity is measured using a microturbidity assay.This is based on the formation of an insoluble precipitate whenhyaluronic acid binds with serum albumin. The activity is measured byincubating hyaluronidase with sodium hyaluronate (hyaluronic acid) for aset period of time (e.g. 10 minutes) and then precipitating theundigested sodium hyaluronate with the addition of acidified serumalbumin. The turbidity of the resulting sample is measured at 640 nmafter an additional development period. The decrease in turbidityresulting from hyaluronidase activity on the sodium hyaluronatesubstrate is a measure of hyaluronidase enzymatic activity. In anotherexample, hyaluronidase activity is measured using a microtiter assay inwhich residual biotinylated hyaluronic acid is measured followingincubation with hyaluronidase (see e.g. Frost and Stern (1997) Anal.Biochem. 251:263-269, U.S. Patent Publication No. 20050260186). The freecarboxyl groups on the glucuronic acid residues of hyaluronic acid arebiotinylated, and the biotinylated hyaluronic acid substrate iscovalently couple to a microtiter plate. Following incubation withhyaluronidase, the residual biotinylated hyaluronic acid substrate isdetected using an avidin-peroxidase reaction, and compared to thatobtained following reaction with hyaluronidase standards of knownactivity. Other assays to measure hyaluronidase activity also are knownin the art and can be used in the methods herein (see e.g. Delpech etal., (1995) Anal. Biochem. 229:35-41; Takahashi et al., (2003) Anal.Biochem. 322:257-263).

Methods for Determining the Etiology of Peri-Orbital Puffiness

The present disclosure provides methods for determining an etiology ofupper and/or lower eyelid puffiness. Such methods comprise examining asubject with squinted eye; and determining if upper and/or lower eyelidpuffiness does not improve, improves, partially improves, or worsens.The etiology of the upper and/or lower eyelid puffiness is determined tobe anterior to the orbicularis oculi muscle if the puffiness does notimprove, the etiology of the upper and/or lower eyelid puffiness isdetermined to be posterior to the orbicularis oculi muscle if thepuffiness improves, the etiology of the upper and/or lower eyelidpuffiness is determined to be anterior and posterior to the orbicularisoculi muscle if the puffiness partially improves, or the puffiness isdetermined to be secondary to hypertrophy of the orbicularis muscle orif the puffiness worsens.

In an embodiment, the subject is in an upright position with head in aFrankfort horizontal plane. In a further embodiment, the methods furthercomprise the step of instructing the subject to squint or tighten theorbicularis oculi muscle.

In an embodiment where the etiology of the upper and/or lower eyelidpuffiness is determined to be anterior to the orbicularis oculi musclethe method further comprises administering a protein havinghyaluronidase activity (e.g., as described herein) into the soft tissueanterior to the orbicularis oculi muscle.

In an embodiment where the etiology of the upper and/or lower eyelidpuffiness is determined to be posterior to the orbicularis oculi muscle,the method further comprises the step of determining if the upper and/orlower eyelid puffiness is secondary to pseudoherniation of upper and/orlower eyelid fat pads, edema of upper and/or lower eyelid fat pads, orupper and/or lower eyelid fat pad pseudoherniation and edema. Suchmethod includes the following steps. First, puffiness of the lowereyelid fat pads are assessed by asking the subject to look straight up,look up and to the right, and look up and to the left. The puffiness ofthe lower eyelid fat pads is due to pseudoherniation of the lower eyelidfat pads and surgery is indicated if the lower eyelid fat pads protrudeand are individually isolated. In contrast, the puffiness is due topseudoherniation and edema of the lower eyelid fat pads if the lowereyelid fat pads protrude and are not individually isolated. Second, aprotein having hyaluronidase activity is then injected into the lowereyelid fat pads to determine the extent of edema of the lower eyelid fatpads. Third, the method may further comprise resecting a portion of thelower eyelid fat pads after assessment of the extent of edema to avoidover resection of the fat pad(s). Next, the puffiness of the uppereyelid fat pads are assessed by asking the subject to look straightdown, look down and to the right, and look down and to the left. Thepuffiness of the upper eyelid fat pads is due to pseudoherniation ofupper eyelid fat pads and surgery is indicated if the upper eyelid fatpads protrude and are individually isolated. In contrast, the puffinessis due to pseudoherniation and edema of upper eyelid fat pads if theupper eyelid fat pads protrude and are not individually isolated.Second, a protein having hyaluronidase activity is then injected intothe upper eyelid fat pads to determine the extent of edema of the eyelidfat pads. Third, the method may further comprise resecting a portion ofthe lower eyelid fat pads after assessment of the extent of edema toavoid over resection of the fat pad(s).

In an embodiment where the etiology of the upper and/or lower eyelidpuffiness is determined to be anterior and posterior to the orbicularisoculi muscle, the method further comprises injecting a protein havinghyaluronidase activity into the upper and/or lower eyelid fat pads.

In an embodiment where the etiology of the upper and/or lower eyelidpuffiness is determined to be anterior and posterior to the orbicularisoculi muscle, and wherein the method further comprises assessing whetherthe puffiness is partially due to pseudoherniation of eyelid fat pads oredema of the fat pads, or eyelid fat pad pseudoherniation and edema.Such method includes the following steps. First, puffiness of the lowereyelid fat pads are assessed by asking the subject to look straight up,look up and to the right, and look up and to the left. The puffiness ofthe lower eyelid fat pads is due to pseudoherniation of the lower eyelidfat pads and surgery is indicated if the lower eyelid fat pads protrudeand are individually isolated. In contrast, the puffiness is due topseudoherniation and edema of the lower eyelid fat pads if the lowereyelid fat pads protrude and are not individually isolated. Second, aprotein having hyaluronidase activity is then injected into the lowereyelid fat pads to determine the extent of edema of the lower eyelid fatpads. Third, the method may further comprise resecting a portion of thelower eyelid fat pads after assessment of the extent of edema to avoidover resection of the fat pad(s). Next, the puffiness of the uppereyelid fat pads are assessed by asking the subject to look straightdown, look down and to the right, and look down and to the left. Thepuffiness of the upper eyelid fat pads is due to pseudoherniation ofupper eyelid fat pads and surgery is indicated if the upper eyelid fatpads protrude and are individually isolated. In contrast, the puffinessis due to pseudoherniation and edema of upper eyelid fat pads if theupper eyelid fat pads protrude and are not individually isolated.Second, a protein having hyaluronidase activity is then injected intothe upper eyelid fat pads to determine the extent of edema of the eyelidfat pads. Third, the method may further comprise resecting a portion ofthe lower eyelid fat pads after assessment of the extent of edema toavoid over resection of the fat pad(s).

The present disclosure also provides methods for determining an etiologyof periorbital puffiness by performing an eyelid squint test (e.g.,asking or having a patient squint their eyes); and observing an impactof a movement of an orbicularis oculi muscle on protrusion of eyelid fatpads, wherein the etiology of the upper and/or lower eyelid puffiness isdetermined to be anterior to the orbicularis oculi muscle if thepuffiness does not improve, wherein the etiology of the upper and/orlower eyelid puffiness is determined to be posterior to the orbicularisoculi muscle if the puffiness improves, wherein the etiology of theupper and/or lower eyelid puffiness is determined to be anterior andposterior to the orbicularis oculi muscle if the puffiness partiallyimproves, or wherein the puffiness is determined to be secondary tohypertrophy of the orbicularis muscle or if the puffiness worsens.

The compositions can be formulated in lyophilized or liquid form. Wherethe compositions are provided in lyophilized form they can bereconstituted just prior to use by an appropriate buffer, for example, asterile saline solution. The compositions can be provided together orseparately. The compositions can be packaged as a kit.

The compositions can be formulated into any suitable pharmaceuticalpreparations for subcutaneous administration such as solutions,suspensions, powders, or sustained release formulations. Typically, thecompositions are formulated into pharmaceutical compositions usingtechniques and procedures well known in the art (see e.g., AnselIntroduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).Pharmaceutically acceptable compositions are prepared in view ofapprovals for a regulatory agency or other agency prepared in accordancewith generally recognized pharmacopeia for use in animals and in humans.The formulation should suit the mode of administration.

Pharmaceutical compositions can include carriers such as a diluent,adjuvant, excipient, or vehicle with which a hyaluronidase or IG isadministered. Examples of suitable pharmaceutical carriers are describedin “Remington's Pharmaceutical Sciences” by E. W. Martin. Suchcompositions will contain a therapeutically effective amount of thecompound, generally in purified form or partially purified form,together with a suitable amount of carrier so as to provide the form forproper administration to the patient. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, and sesame oil. Water is a typical carrierwhen the pharmaceutical composition is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions also can beemployed as liquid carriers, particularly for injectable solutions.Compositions can contain along with an active ingredient: a diluent suchas lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; alubricant, such as magnesium stearate, calcium stearate and talc; and abinder such as starch, natural gums, such as gum acaciagelatin, glucose,molasses, polyvinylpyrrolidine, celluloses and derivatives thereof,povidone, crospovidones and other such binders known to those of skillin the art. Suitable pharmaceutical excipients include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, and ethanol. A composition, ifdesired, also can contain minor amounts of wetting or emulsifyingagents, or pH buffering agents, for example, acetate, sodium citrate,cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodiumacetate, triethanolamine oleate, and other such agents.

Pharmaceutically therapeutically active compounds and derivativesthereof are typically formulated and administered in unit dosage formsor multiple dosage forms. Each unit dose contains a predeterminedquantity of therapeutically active compound sufficient to produce thedesired therapeutic effect, in association with the requiredpharmaceutical carrier, vehicle or diluent. Examples of unit dose formsinclude ampoules and syringes and individually packaged tablets orcapsules. Unit dose forms can be administered in fractions or multiplesthereof. A multiple dose form is a plurality of identical unit dosageforms packaged in a single container to be administered in segregatedunit dose form. Examples of multiple dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit doses that are not segregated inpackaging. Generally, dosage forms or compositions containing activeingredient in the range of 0.005% to 100% with the balance made up fromnon-toxic carrier can be prepared.

Compositions provided herein typically are formulated for administrationby subcutaneous route, although other routes of administration arecontemplated, such as any route known to those of skill in the art.Formulations suited for such routes are known to one of skill in theart. Administration can be local, topical or systemic depending upon thelocus of treatment. Local administration to an area in need of treatmentcan be achieved by, for example, but not limited to, local infusionduring surgery, topical application, transdermal patch, or by injection.Compositions also can be administered with other biologically activeagents, either sequentially, intermittently or in the same composition.Thus, in one example, local administration can be achieved by injection,such as from a syringe or other article of manufacture containing aninjection device such as a needle or an injection device containingmultiple needles. In another example, local administration can beachieved by infusion, which can be facilitated by the use of a pump orother similar device, or by a transdermal patch. Pharmaceuticalcompositions can be formulated in dosage forms appropriate for eachroute of administration.

Subcutaneous administration, generally characterized by injection orinfusion, is contemplated herein. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. The pharmaceutical compositions maycontain other minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents, stabilizers,solubility enhancers, and other such agents, such as for example, sodiumacetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see, e.g., U.S. Pat. No.3,710,795) is also contemplated herein. The percentage of activecompound contained in such compositions is highly dependent on thespecific nature thereof, as well as the activity of the compound and theneeds of the subject.

Injectables are designed for local and systemic administration. Forpurposes herein, local administration is desired for directadministration to the affected area. The solutions may be either aqueousor nonaqueous.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers, which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose.

Buffers include phosphate and citrate. Antioxidants include sodiumbisulfate. Local anesthetics include procaine hydrochloride. Suspendingand dispersing agents include sodium carboxymethylcellulose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEENs 80). A sequestering or chelatingagent of metal ions include EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art. The unit-doseparenteral preparations are packaged in an ampoule, a vial or a syringewith a needle. The volume of liquid solution or reconstituted powderpreparation, containing the pharmaceutically active compound, is afunction of the disease to be treated and the particular article ofmanufacture chosen for package.

In one example, a pharmaceutical preparation can be in liquid form, forexample, solutions, syrups or suspensions. If provided in liquid form,the pharmaceutical preparations can be provided as a concentratedpreparation to be diluted to a therapeutically effective concentrationbefore use. Such liquid preparations can be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). In another example, pharmaceutical preparations can bepresented in lyophilized form for reconstitution with water or othersuitable vehicle before use.

Administration methods can be employed to decrease the exposure of thehyaluronidase to degradative processes, such as proteolytic degradationand immunological intervention via antigenic and immunogenic responses.Examples of such methods include local administration at the site oftreatment. Pegylation of therapeutics has been reported to increaseresistance to proteolysis, increase plasma half-life, and decreaseantigenicity and immunogenicity. Examples of pegylation methodologiesare known in the art (see for example, Lu and Felix, Int. J. PeptideProtein Res., 43: 127-138, 1994; Lu and Felix, Peptide Res., 6: 142-6,1993; Felix et al., Int. J. Peptide Res., 46: 253-64, 1995; Benhar etal., J. Biol. Chem., 269: 13398-404, 1994; Brumeanu et al., J Immunol.,154: 3088-95, 1995; see also, Caliceti et al. (2003) Adv. Drug Deliv.Rev. 55(10):1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt2):3S-8S). Pegylation also can be used in the delivery of nucleic acidmolecules in vivo. For example, pegylation of adenovirus can increasestability and gene transfer (see, e.g., Cheng et al. (2003) Pharm. Res.20(9): 1444-2. Dosage and Administration.

Typically, a therapeutically effective dose is at or about 1 Unit to100,000 Units of a soluble hyaluronidase. For example, solublehyaluronidase can be administered subcutaneously at or about 10 units,20 Units, 50 Units, 100 Units, 200 Units, 500 Units, 1000 Units, 2000Units, 5000 Units, 10,000 Units, 30,000 Units, 40,000 Units, 50,000Units, 60,000 Units, 70,000 Units, 80,000 Units, 90,000 Units, 100,000Units or more. Typically, volumes of injections or infusions ofhyaluronidase contemplated herein are from at or about 0.1 ml, 0.2 ml,0.3 ml, 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or more. The hyaluronidase can beprovided as a stock solution at or about 50 U/ml, 100 U/ml, 150 U/ml,200 U/ml, 400 U/ml or 500 U/ml or can be provided in a more concentratedform, for example at or about 1000 U/ml, 1500 Units/ml, 2000 U/ml, 4000U/ml or 5000 U/ml for use directly or for dilution to the effectiveconcentration prior to use.

The actual amount of the hyaluronidase to be administered in any givencase will be determined by a physician or other skilled person takinginto account the relevant circumstances, such as the amount of edema inthe tissues, the desired reduction in the puffiness, the potential fatreduction, the age and weight of the patient, the patient's generalphysical condition, the cause of the condition, and the route ofadministration.

Other therapeutically efficient amounts of a hyaluronidase will beapparent to a skilled person upon a reading of the present disclosure.For example, a skilled person can determine the maximum safe dosage forhealthy subjects based on the dosages used in animal studies by routinemethods (see, e.g. Dept. of Health and Human Services “Guidance ForIndustry: Estimating the Maximum Safe Starting Dose in Initial ClinicalTrials for Therapeutics in Adult Healthy Volunteers”), and thenadminister to subjects in need thereof various dosages below the maximumsafe dosage by routine methods and experimentation until a dosage whichresults in a desirable effect (e.g. reduction in the extent ofperi-orbital puffiness, festoons, or malar puffiness due to edema) isreached.

The therapeutically efficient amount of a hyaluronidase can be presentin a formulation (e.g. for topical administration) at between about 0.01and about 5% (w/v). In some embodiments, the therapeutically effectiveamount in the formulation can be from about 0.01 to about 1%, about 0.01to about 2%, about 0.01 to about 3%, and about 0.01 to about 4%. Inother embodiments, the therapeutically effective amount in theformulation can be from about 0.01 to about 1%, about 1 to about 2%,about 2 to about 3%, about 3 to about 4%, about 4 to about 5%.

In other embodiments, the therapeutically effective amount of ahyaluronidase in the formulation can be from about 0.01 to about 0.06%,about 0.06 to about 0.11%, about 0.11 to about 0.16%, about 0.16 toabout 0.21%, about 0.21 to about 0.26%, about 0.26 to about 0.31%, about0.31 to about 0.36%, about 0.36 to about 0.41%, about 0.41 to about0.46%, about 0.46 to about 0.51%, about 0.51 to about 0.56%, about 0.56to about 0.61%, about 0.61 to about 0.66%, about 0.66 to about 0.71%,about 0.71 to about 0.76%, about 0.76 to about 0.81%, about 0.81 toabout 0.86%, about 0.86 to about 0.91%, about 0.91 to about 0.96%, about0.96 to about 1.01%, about 1.01 to about 1.06%, about 1.06 to about1.11%, about 1.11 to about 1.16%, about 1.16 to about 1.21%, about 1.21to about 1.26%, about 1.26 to about 1.31%, about 1.31 to about 1.36%,about 1.36 to about 1.41%, about 1.41 to about 1.46%, about 1.46 toabout 1.51%, about 1.51 to about 1.56%, about 1.56 to about 1.61%, about1.61 to about 1.66%, about 1.66 to about 1.71%, about 1.71 to about1.76%, about 1.76 to about 1.81%, about 1.81 to about 1.86%, about 1.86to about 1.91%, about 1.91 to about 1.96%, about 1.96 to about 2.01%,about 2.01 to about 2.06%, about 2.06 to about 2.11%, about 2.11 toabout 2.16%, about 2.16 to about 2.21%, about 2.21 to about 2.26%, about2.26 to about 2.31%, about 2.31 to about 2.36%, about 2.36 to about2.41%, about 2.41 to about 2.46%, about 2.46 to about 2.51%, about 2.51to about 2.56%, about 2.56 to about 2.61%, about 2.61 to about 2.66%,about 2.66 to about 2.71%, about 2.71 to about 2.76%, about 2.76 toabout 2.81%, about 2.81 to about 2.86%, about 2.86 to about 2.91%, about2.91 to about 2.96%, about 2.96 to about 3.01%, about 3.01 to about3.06%, about 3.06 to about 3.11%, about 3.11 to about 3.16%, about 3.16to about 3.21%, about 3.21 to about 3.26%, about 3.26 to about 3.31%,about 3.31 to about 3.36%, about 3.36 to about 3.41%, about 3.41 toabout 3.46%, about 3.46 to about 3.51%, about 3.51 to about 3.56%, about3.56 to about 3.61%, about 3.61 to about 3.66%, about 3.66 to about3.71%, about 3.71 to about 3.76%, about 3.76 to about 3.81%, about 3.81to about 3.86%, about 3.86 to about 3.91%, about 3.91 to about 3.96%,about 3.96 to about 4.01%, about 4.01 to about 4.06%, about 4.06 toabout 4.11%, about 4.11 to about 4.16%, about 4.16 to about 4.21%, about4.21 to about 4.26%, about 4.26 to about 4.31%, about 4.31 to about4.36%, about 4.36 to about 4.41%, about 4.41 to about 4.46%, about 4.46to about 4.51%, about 4.51 to about 4.56%, about 4.56 to about 4.61%,about 4.61 to about 4.66%, about 4.66 to about 4.71%, about 4.71 toabout 4.76%, about 4.76 to about 4.81%, about 4.81 to about 4.86%, about4.86 to about 4.91%, about 4.91 to about 4.96%, and about 4.96 to about5% (w/v).

The therapeutically effective amount can be administered according to adosing frequency that is identifiable to a skilled person during a timeperiod that is also identifiable to a skilled person. The term “dosingfrequency” as used herein, refers to the number of times the compoundsdescribed herein are administered to a subject. Exemplary dosingfrequencies include administering the effective amount at discrete timesduring a day such as, for example, once a day (QD), twice a day (BID),three times a day (TID), four times a day (QID), and others identifiableto a skilled person. Other exemplary dosing frequencies includecontinuous dosing, for example by intravenous infusion, use of a drugpump, use of a transdermal patch, or other methods of continuous dosingidentifiable to a skilled person.

The therapeutically effective amount can be administered at a desireddosing frequency for a time period identifiable to a skilled person. Forexample, a therapeutically effective can be administered once or twice aday (or at another dosing frequency identifiable to a skilled person)for a set period of time (e.g. seven to fourteen days, two to fourweeks, one to six months, or for another time period identifiable to askilled person). As another example, a therapeutically effective amountcan be administered once or twice a day (or at another dosing frequencyidentifiable to a skilled person) for a non-predetermined period oftime. A skilled person can determine at various points during the periodof time if the administration of the effective amount is to becontinued.

Pharmaceutical compositions of hyaluronidase can be packaged as articlesof manufacture containing packaging material, a pharmaceuticalcomposition which is effective for treating puffiness, and a label thatindicates that the composition is to be used for treating puffiness.Exemplary of articles of manufacture are containers including singlechamber and dual chamber containers. The containers include, but are notlimited to, tubes, bottles and syringes. The containers can furtherinclude a needle for subcutaneous administration.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, for example, U.S. Pat.Nos. 5,323,907, 5,033,252 and 5,052,558, each of which is incorporatedherein in its entirety. Examples of pharmaceutical packaging materialsinclude, but are not limited to, blister packs, bottles, tubes,inhalers, pumps, bags, vials, containers, syringes, bottles, and anypackaging material suitable for a selected formulation and intended modeof administration and treatment.

A hyaluronidase composition may optionally comprise an anesthetic agent.An anesthetic agent may be a local anesthetic agent, including ananesthetic agent that causes a reversible local anesthesia or a loss ofnociception, such as, e.g., aminoamide local anesthetics and aminoesterlocal anesthetics. Non-limiting examples of anesthetic agents mayinclude lidocaine, ambucaine, amolanone, amylocaine, benoxinate,benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben,butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine,cocaethylene, cyclomethycaine, dibucaine, dimethisoquin, dimethocaine,diperodon, dicyclomine, ecgonidine, ecgonine, ethyl chloride,etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine,hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate,levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methylchloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine,parethoxycaine, phenacaine, phenol, piperocaine, piridocaine,polidocanol, pramoxine, prilocaine, procaine, propanocaine,proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine,ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine,zolamine, combinations thereof, and salts thereof. Non-limiting examplesof aminoester local anesthetics include procaine, chloroprocaine,cocaine, cyclomethycaine, dimethocaine (larocaine), propoxycaine,procaine (novocaine), proparacaine, tetracaine (amethocaine).Non-limiting examples of aminoamide local anesthetics include articaine,bupivacaine, cinchocaine (dibucaine), etidocaine, levobupivacaine,lidocaine (lignocaine), mepivacaine, piperocaine, prilocaine,ropivacaine, trimecaine, or a combination thereof.

The amount of an anesthetic agent included may be an amount effective toreduce pain experienced by an individual upon administration of thecomposition, such as about 0.1%, about 0.2%, about 0.3%, about 0.4%,about 0.5%, about 0.6%, about 0.7%, about 0.8% about 0.9%, about 1.0%,about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%,about 8.0%, about 9.0%, about 10%, at least about 0.1%, at least about0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, atleast about 0.6%, at least about 0.7%, at least about 0.8% at leastabout 0.9%, at least about 1.0%, at least about 2.0%, at least about3.0%, at least about 4.0%, at least about 5.0%, at least about 6.0%, atleast about 7.0%, at least about 8.0%, at least about 9.0%, at leastabout 10%, at most about 0.1%, at most about 0.2%, at most about 0.3%,at most about 0.4%, at most about 0.5%, at most about 0.6%, at mostabout 0.7%, at most about 0.8% at most about 0.9%, at most about 1.0%,at most about 2.0%, at most about 3.0%, at most about 4.0%, at mostabout 5.0%, at most about 6.0%, at most about 7.0%, at most about 8.0%,at most about 9.0%, at most about 10%, about 0.1% to about 0.5%, about0.1% to about 1.0%, about 0.1% to about 2.0%, about 0.1% to about 3.0%,about 0.1% to about 4.0%, about 0.1% to about 5.0%, about 0.2% to about0.9%, about 0.2% to about 1.0%, about 0.2% to about 2.0%, about 0.5% toabout 1.0%, or about 0.5% to about 2.0%.

Some hyaluronidase compositions may comprise lidocaine, in free base orsalt form (e.g. lidocaine HCl) in an amount of about 0.05% w/w to about1% w/w; about 0.1% w/w to about 0.5% w/w, or about 0.3% w/w.

Additionally, compositions of hyaluronidase may have aphysiologically-acceptable osmolarity, e.g., about 100 mOsm/L, about 150mOsm/L, about 200 mOsm/L, about 250 mOsm/L, about 300 mOsm/L, about 350mOsm/L, about 400 mOsm/L, about 450 mOsm/L, about 500 mOsm/L, at leastabout 100 mOsm/L, at least about 150 mOsm/L, at least about 200 mOsm/L,at least about 250 mOsm/L, at most about 300 mOsm/L, at most about 350mOsm/L, at most about 400 mOsm/L, at most about 450 mOsm/L, at mostabout 500 mOsm/L, about 100 mOsm/L to about 500 mOsm/L, about 200 mOsm/Lto about 500 mOsm/L, about 200 mOsm/L to about 400 mOsm/L, about 300mOsm/L to about 400 mOsm/L, about 270 mOsm/L to about 390 mOsm/L, about225 mOsm/L to about 350 mOsm/L, about 250 mOsm/L to about 325 mOsm/L,about 275 mOsm/L to about 300 mOsm/L, or about 285 mOsm/L to about 290mOsm/L. Osmolality agents may be used to adjust osmolality. Examplesinclude, but are not limited to, salts such as, e.g., sodium chlorideand potassium chloride; and glycerin.

In some embodiments, a composition comprising hyaluronidase isinjectable through a needle of, e.g., about 27 gauge; about 30 gauge;about 32 gauge; about 22 gauge or smaller; about 27 gauge or smaller;about 30 gauge or smaller; about 32 gauge or smaller; about 22 gauge toabout 35 gauge; about 22 gauge to about 34 gauge; about 22 gauge toabout 33 gauge; about 22 gauge to about 32 gauge; about 22 gauge toabout 27 gauge; or about 27 gauge to about 32 gauge.

An hyaluronidase composition may be substantially stable at roomtemperature, e.g., for about 3 months, about 6 months, about 9 months,about 12 months, about 15 months, about 18 months, about 21 months,about 24 months, about 27 months, about 30 months, about 33 months,about 36 months, at least about 3 months, at least about 6 months, atleast about 9 months, at least about 12 months, at least about 15months, at least about 18 months, at least about 21 months, at leastabout 24 months, at least about 27 months, at least about 30 months, atleast about 33 months, at least about 36 months, about 3 months to about12 months, about 3 months to about 18 months, about 3 months to about 24months, about 3 months to about 30 months, about 3 months to about 36months, about 6 months to about 12 months, about 6 months to about 18months, about 6 months to about 24 months, about 6 months to about 30months, about 6 months to about 36 months, about 9 months to about 12months, about 9 months to about 18 months, about 9 months to about 24months, about 9 months to about 30 months, about 9 months to about 36months, about 12 months to about 18 months, about 12 months to about 24months, about 12 months to about 30 months, about 12 months to about 36months, about 18 months to about 24 months, about 18 months to about 30months, or about 18 months to about 36 months.

A hyaluronidase may be injected at between about 2 and about 5 sites. Inan embodiment, the hyaluronidase is injected at between about 5 andabout 10 sites. In an embodiment, the hyaluronidase is injected atbetween about 10 to about 30 sites. In an embodiment, the hyaluronidaseis injected at between about 10 to about 50 sites. At least two of thesites can be separated by a distance of approximately 100 microns toabout 5,000 microns. In an embodiment, the distance between injectionsites is about 400 to about 600 microns. In an embodiment, the distancebetween injections sites is about 100 to about 200 microns, about 200 toabout 300 microns, about 300 to about 400 microns, about 400 to about500 microns, about 500 to about 600 microns, about 600 to about 700microns, about 700 to about 800 microns, about 800 to about 900 microns,or about 900 to about 1,000 microns. In an embodiment, the distancebetween injection sites is about 1,000 to about 2,000 microns, about2,000 to about 3,000 microns, about 3,000 to about 4,000 microns, orabout 4,000 to about 5,000 microns.

In an embodiment, the protein having hyaluronidase activity is injectedinto a lateral eyelid fat pad, a medial eyelid fat pad, and/or a centraleyelid fat pad. In a further embodiment, 1 to 15 injections, preferably1 to 3 injections, are made into each of the lateral eyelid fat pad,medial eyelid fat pad, and/or central eyelid fat pad. Preferably, theinjections into the medial eyelid fat pad are made at a depth of betweenabout 3 to about 10 mm, more preferably about 5 to about 8 mm.Additionally, the injections into the central eyelid fat pad arepreferably made at a depth of between about 3 to about 12 mm, morepreferably about 5 to about 10 mm. Furthermore, the injections into thelateral eyelid fats pad are preferably made at a depth of between about3 to about 11 mm, more preferably about 5 to about 8 mm.

In an embodiment, the protein having hyaluronidase activity is injectedinto a malar region. In a further embodiment, 1 to 15 injections,preferably 3 to 7 injections, are made into the malar region.Preferably, the injections into the malar region are made at a depth ofbetween about 2 to about 13 mm, more preferably about 5 to about 10 mm.

In some embodiments of any of the aforementioned methods, thehyaluronidase is administered once. In some embodiments of any of theaforementioned methods, administration of an initial dose thehyaluronidase is followed by the administration of one or moresubsequent doses of the hyaluronidase. Examples of dosing regimens(e.g., an interval between the first dose and one or more subsequentdoses) that can be used in the methods of the disclosure include aninterval of about once every week to about once every 12 months, aninterval of about once every two weeks to about once every 6 months, aninterval of about once every month to about once every 6 months, aninterval of about once every month to about once every 3 months, or aninterval of about once every 3 months to about once every 6 months. Insome embodiments, administration is monthly, every two months, everythree months, every four months, every five months, every six months, orupon disease recurrence.

The present disclosure is further illustrated by the following examples,which should not be construed as limiting in any way. The materials andmethods as used in the following experimental examples are describedbelow.

EXAMPLES Example 1: Methods for Determining the Etiology of Pen-OrbitalPuffiness

A 43 year old female patient presented with periorbital puffiness of theperiorbital region in both their right and left eye. The patient wasseated with her head in the Frankfort horizontal plane and instructed tosquint both of her eyes. Next, it was determined if upper and/or lowereyelid puffiness did not improve, improved, partially improved, orworsened. The puffiness of the patient's left eyelid did not improve andthus the etiology of the puffiness was determined to be anterior to theorbicularis oculi muscle. Further, the puffiness of the patient's righteyelid partially improved and thus the etiology of the puffiness wasdetermined to be anterior and posterior to the orbicularis oculi muscle.

Example 2: Treatment of Periorbital Puffiness Due to Periorbital EdemaUsing HYLENEX

Four patients (Patients A-D) with periorbital puffiness due to edema (asidentified in Example 1) of the eyelid fat pads were scored as a Grade1E, 2E, 3E, or 3+E on the Periorbital Fullness Assessment Scale. A scorewas given to the upper and lower eyelids in the patient's left and righteye. Patient A's eyes were scored on the PEFAS scale as a L 0/3E (lefteye; upper eyelid over lower eyelid) and a R 0/3E (right eye; uppereyelid over lower eyelid). Patient B was scored as a L 2E/2E and a R2E/2E. Patient C was scored as a L 1/3E and a R 0/3E and Patient D wasscored as a L 2E/3+E and a R 2E/3+E.

The patients were subsequently treated with HYLENEX. Briefly, oneinjection of 30 U HYLENEX was made per periorbital fat pad exhibitingedema and optionally four injections were administered in theperiorbital soft tissues using the same dosage. The injections wereperformed using a 0.5 ml syringe having a 31-gauge needle insertedparallel to the globe, angling away from the globe, to avoid injury tothe globe. Specifically, three injections were made into the medialeyelid fat pad, two into the central eyelid fat pad, and three into thelateral eyelid fat pad at a depth of about 6 mm, 8 mm, and 6 mm,respectively. Additionally Patients B and C received 3 to 5 injectionsin the periorbital soft tissues (e.g., the malar region) at a depth ofbetween about 5 to about 10 mm.

Each patient exhibited an improvement in the extent of periorbitalpuffiness within the first 15-60 minutes after the injections. Aftertreatment, Patient C was followed for 1 month, Patient D was followedfor 2 months, and Patients A and B were followed for 3 months. Eachpatient exhibited a marked reduction in per-orbital (eye) puffiness ascompared to the periorbital puffiness that exhibited pre-treatment (see,FIGS. 1-5). The patients were followed for up to 6 months and stillexhibit a reduction in periorbital puffiness measured pre-treatmentwithout any adverse effects.

After treatment, the patient's eyes were scored on the PEFAS scale.Patient A's eyes were scored on the PFAS scale as a L 0/0 (left eye;upper eyelid over lower eyelid) and a R 0/0E (right eye; upper eyelidover lower eyelid). Patient B was scored as a L 2E/1 E and a R 2E/1.Patient C was scored as a L 1E/1E and a R 0/1 and Patient D was scoredas a L 2E/0E and a R 2E/0. Thus, each patient demonstrated animprovement in peri-orbital edema by a decrease in the grade assigned tothe periorbital puffiness assigned to both their left and right eyes.

Moreover, an additional 12 patients that exhibit periorbital puffinesswere treated according to the regimen described above. Each patient wasgiven an initial Subjective Patient Score of 10. The Subjective PatientScore is based on a scale of 1 to 10 with 10 being the subject'sobserved starting amount of periorbital puffiness and 0 being nopuffiness. Each of the patients scored their puffiness between as a 3 or4 at 2 weeks, 3 months, and 6 months after their initial treatment.

Embodiments

Embodiment 1: A method for determining an etiology of upper and/or lowereyelid puffiness, the method comprising:

-   -   a. examining a subject with squinted eyes; and    -   b. determining if upper and/or lower eyelid puffiness does not        improve, improves, partially improves, or worsens,

wherein the etiology of the upper and/or lower eyelid puffiness isdetermined to be anterior to an orbicularis oculi muscle if the upperand/or lower eyelid puffiness does not improve,

wherein the etiology of the upper and/or lower eyelid puffiness isdetermined to be posterior to the orbicularis oculi muscle if the upperand/or lower eyelid puffiness improves,

wherein the etiology of the upper and/or lower eyelid puffiness isdetermined to be anterior and posterior to the orbicularis oculi muscleif the upper and/or lower eyelid puffiness partially improves, or

wherein the puffiness is determined to be secondary to hypertrophy ofthe orbicularis muscle or if the upper and/or lower eyelid puffinessworsens.

Embodiment 2: The method of embodiment 1, wherein the subject is seatedin an upright position with head positioned in a Frankfort horizontalplane.

Embodiment 3: The method of embodiment 1 further comprising the step ofinstructing the subject to squint or tighten the orbicularis oculimuscle.

Embodiment 4: The method of embodiment 1, wherein the etiology of theupper and/or lower eyelid puffiness is determined to be anterior to theorbicularis oculi muscle, and wherein the method further comprisesadministering a protein having hyaluronidase activity into the softtissue anterior to the orbicularis oculi muscle.

Embodiment 5: The method of embodiment 1, wherein the etiology of theupper and/or lower eyelid puffiness is determined to be posterior to theorbicularis oculi muscle, and wherein the method further comprises thestep of determining if the upper and/or lower eyelid puffiness issecondary to pseudoherniation of upper and/or lower eyelid fat pads,edema of upper and/or lower eyelid fat pads, or upper and/or lowereyelid fat pad pseudoherniation and edema.

Embodiment 6: The method of embodiment 5, wherein the puffiness of thelower eyelid fat pads are assessed by asking the subject to lookstraight up, look up and to the right, and look up and to the left.

Embodiment 7: The method of embodiment 6, wherein the puffiness of thelower eyelid fat pads is due to pseudoherniation of the lower eyelid fatpads and surgery is indicated if the lower eyelid fat pads protrude andare individually isolated.

Embodiment 8: The method of embodiment 5, wherein the puffiness is dueto pseudoherniation and edema of the lower eyelid fat pads if the lowereyelid fat pads protrude and are not individually isolated.

Embodiment 9: The method of embodiment 8, wherein a protein havinghyaluronidase activity is injected into the lower eyelid fat pads todetermine the extent of edema of the lower eyelid fat pads.

Embodiment 10: The method of embodiment 9 further comprising resecting aportion of the lower eyelid fat pads.

Embodiment 11: The method of embodiment 5, wherein the puffiness of theupper eyelid fat pads are assessed by asking the subject to lookstraight down, look down and to the right, and look down and to theleft.

Embodiment 12: The method of embodiment 11, wherein the puffiness of theupper eyelid fat pads is due to pseudoherniation of upper eyelid fatpads and surgery is indicated if the upper eyelid fat pads protrude andare individually isolated.

Embodiment 13: The method of embodiment 11, wherein the puffiness is dueto pseudoherniation and edema of upper eyelid fat pads if the uppereyelid fat pads protrude and are not individually isolated.

Embodiment 14: The method of embodiment 13, wherein a protein havinghyaluronidase activity is injected into the upper eyelid fat pads todetermine the extent of edema of the eyelid fat pads.

Embodiment 15: The method of embodiment 14 further comprising resectinga portion of the upper eyelid fat pads.

Embodiment 16: The method of embodiment 1, wherein the etiology of theupper and/or lower eyelid puffiness is determined to be anterior andposterior to the orbicularis oculi muscle, and wherein the methodfurther comprises injecting a protein having hyaluronidase activity intothe upper and/or lower eyelid fat pads.

Embodiment 17: The method of embodiment 1, wherein the etiology of theupper and/or lower eyelid puffiness is determined to be anterior andposterior to the orbicularis oculi muscle, and wherein the methodfurther comprises assessing whether the puffiness is partially due topseudoherniation of eyelid fat pads or edema of the fat pads, or eyelidfat pad pseudoherniation and edema.

Embodiment 18: The method of embodiment 17, wherein the puffiness of thelower eyelid fat pads are assessed by asking the subject to lookstraight up, look up and to the right, and look up and to the left.

Embodiment 19: The method of embodiment 18, wherein the puffiness is dueto pseudoherniation of the lower eyelid fat pads and surgery isindicated if the lower eyelid fat pads protrude and are individuallyisolated.

Embodiment 20: The method of embodiment 18, wherein the puffiness is dueto pseudoherniation and edema of the lower eyelid fat pads if the lowereyelid fat pads protrude and are not individually isolated.

Embodiment 21: The method of embodiment 20, wherein a protein havinghyaluronidase activity is injected into the lower eyelid fat pads todetermine the extent of edema of the eyelid fat pads.

Embodiment 22: The method of embodiment 21 further comprising resectinga portion of the lower eyelid fat pads.

Embodiment 23: The method of embodiment 17, wherein the puffiness of theupper eyelid fat pads are assessed by asking the subject to lookstraight down, look down and to the right, and look down and to theleft.

Embodiment 24: The method of embodiment 23, wherein the puffiness of theupper eyelid fat pads is due to pseudoherniation of the upper eyelid fatpads and surgery is indicated if the upper eyelid fat pads protrude andare individually isolated.

Embodiment 25: The method of embodiment 23, wherein the puffiness of theupper eyelid fat pads is due to pseudoherniation and edema of the uppereyelid fat pads if the upper eyelid fat pads protrude and are notindividually isolated.

Embodiment 26: The method of embodiment 25, wherein a protein havinghyaluronidase activity is injected into the upper eyelid fat pads todetermine the extent of edema of the upper eyelid fat pads.

Embodiment 27: The method of embodiment 26 further comprising resectinga portion of the upper eyelid fat pads.

Embodiment 28: The method of embodiment 1, wherein a neuromodulator isindicated if the puffiness is determined to be secondary to hypertrophyof the orbicularis muscle or if the puffiness worsens.

Embodiment 29: A method for determining an etiology of periorbitalpuffiness, the method comprising:

-   -   performing an eyelid squint test; and    -   observing an impact of a movement of an orbicularis oculi muscle        on protrusion of eyelid fat pads,

wherein the etiology of the upper and/or lower eyelid puffiness isdetermined to be anterior to the orbicularis oculi muscle if thepuffiness does not improve,

wherein the etiology of the upper and/or lower eyelid puffiness isdetermined to be posterior to the orbicularis oculi muscle if thepuffiness improves,

wherein the etiology of the upper and/or lower eyelid puffiness isdetermined to be anterior and posterior to the orbicularis oculi muscleif the puffiness partially improves, or

wherein the puffiness is determined to be secondary to hypertrophy ofthe orbicularis muscle or if the puffiness worsens.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group can be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the disclosureto be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein can be further limited in theclaims using “consisting of” or “consisting essentially of” language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the disclosure so claimed areinherently or expressly described and enabled herein.

It is to be understood that the embodiments of the disclosure disclosedherein are illustrative of the principles of the present disclosure.Other modifications that can be employed are within the scope of thedisclosure. Thus, by way of example, but not of limitation, alternativeconfigurations of the present disclosure can be utilized in accordancewith the teachings herein. Accordingly, the present disclosure is notlimited to that precisely as shown and described.

While the present disclosure has been described and illustrated hereinby references to various specific materials, procedures and examples, itis understood that the disclosure is not restricted to the particularcombinations of materials and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplary only, with thetrue scope and spirit of the disclosure being indicated by the followingclaims. All references, patents, and patent applications referred to inthis application are herein incorporated by reference in their entirety.

1. A method for determining an etiology of upper and/or lower eyelidperiorbital puffiness in a subject and treating the subject, the methodcomprising: a. examining the upper and/or lower eyelid periorbitalpuffiness in the subject with and without squinted eyes; b. determiningthat the upper and/or lower eyelid periorbital puffiness is anterior toan orbicularis oculi muscle where the upper and/or lower eyelidperiorbital puffiness does not improve with squinted eyes; and c.administering a protein having hyaluronidase activity into the softtissue anterior to the orbicularis oculi muscle of the subject, whereinthe upper and/or lower eyelid periorbital puffiness is reduced in thesubject for about 2 months or more.
 2. The method of claim 1, whereinthe subject is seated in an upright position with head positioned in aFrankfort horizontal plane.
 3. The method of claim 1 further comprisingthe step of instructing the subject to squint or tighten the orbicularisoculi muscle before the step of examining the subject with squintedeyes.
 4. (canceled)
 5. (canceled)
 6. The method of claim 1, wherein theperiorbital puffiness of the lower eyelid fat pads are assessed byasking the subject to look straight up, look up and to the right, andlook up and to the left. 7-9. (canceled)
 10. The method of claim 1,further comprising resecting a portion of the lower eyelid fat pads. 11.The method of claim 1, wherein the periorbital puffiness of the uppereyelid fat pads are assessed by asking the subject to look straightdown, look down and to the right, and look down and to the left. 12-29.(canceled)